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Supplementary Materials
Supplementary Methods:
Patients: This institutional review board (IRB) approved analysis included retrospective examination of
patient charts and utilization of pretransplant and posttransplant peripheral blood (PB) and bone
marrow (BM) samples. PB and BM samples are routinely stored in the pathology department for clinical
chimerism studies and their use for this analysis was approved by the IRB. In addition PB and BM
samples were collected at the time of relapse.
Flow cytometry: A fluorescence-activated cell sorter was utilized to isolate leukemic blasts from the PB
and BM samples. PB and BM mononuclear cells were stained with either of the following combinations
of monoclonal antibodies: anti CD45 – PE and anti-HLA-DR – FITC (Beckton Dickinson Biosciences) or
anti-CD3 – APC and CD33 – ef450 (eBioscience). Cells were sorted with a FACSAria II cell sorter (Beckton
Dickinson) into T cells (SSClo CD45hi CD3+) and leukemic blasts, defined as SSCint CD45lo HLA-DR+ and SSCint
CD45lo CD33+ for recipient 1 and recipient 2, respectively.
Reverse sequence specific oligonucleotide (rSSO) analysis: Reverse sequence specific oligonucleotide
(rSSO) probes multianalyte bead method (LABType, One Lambda a ThermoFisher Scientific Brand) was
performed by rSSO hybridization on microbeads arrays (One Lambda HD kits, Canoga Park, CA, USA).
Mixing experiments were performed to define more precisely a cutoff limit for the detection of a
heterozygous DNA present in a fraction of the cells by the luminex technology.
Melting Curve Analysis: Melting curve analysis (reagents PowerSYBR Green Master Mix from
ThermoFischer Scientific) was performed on genomic DNA of PB and BM specimens. Primer sequences
for recipient 1 were HLA-A*26 forward GGAGTATTGGGACCGGAAC at (exon 2 nucleotides 243-261) and
reverse were CAGGGTCCCCAGGTTCG (Exon 2 nucleotides 299-315). For recipient 2 HLA-A*03 forward
primer sequences were ATGGCGGCTCAGATCACCA (exon 3 nucleotides 484-502) and reverse
CACTCCACGCACGTGCCA (exon 3 nucleotides 555-572). The sensitivities of the assays were determined
by serial dilutions of the recipient’s pretransplant samples. DNA concentrations for the dilution studies
were determined by Qubit DNA fluorometric quantitation (ThermoFisher Scientific) and 6.6pg of DNA
was considered a single cell equivalent. Results were compared with those obtained from the recipient
and donor prior to transplantation, which served as reference controls.
Single nucleotide polymorphism (SNP) array analysis: The SNP array was performed on DNA obtained
from BM specimens, using HumanCytoSNP12 Beadchip containing over 300K markers (IlluminaInc. USA).
The B allele frequency (BAF) and Log R ratio (LRR) data were analyzed using Illumina KaryoStudio
software version 2.0 and copy number variation (CNV) partition 2.4.4.0. BAF represents the frequency of
B alleles at a given SNP. LRR represents the ratio between the observed and the expected probe
intensity, thus indicating copy number.